Microorganisms commonly encounter threatening changes in their environments. These changes include depletion of nutrients, shifts in pH, temperature and pressure, and sharing habitats with invading organisms. A commonly encountered stress of animal-associated bacteria and one of importance to a study of infectious diseases is that induced through the transfer of cultures from their propagation media into different media. This stress is especially pronounced when the bacteria are transferred to a media of neutral pH with altered or reduced levels of nutrients, such as when exogenous bacteria enter an animal or when endogenous bacteria penetrate sterile zones and tissues from their non-sterile habitats. During growth, bacteria form H+ which accumulate in both the cell and the media.
When encountering normal and naturally-occurring stresses, bacteria release products known as stress response factors, (SRFs). These SRFs include polymers of nucleic acids and their partial and complete hydrolysates including a mixture of about a dozen oligoribonucleotides (ORNs) ranging from 1–30 nucleotides, which are resistant to further hydrolysis by RNAse.
The present invention has found that a series of mild stresses, mimicking those commonly experienced when bacterial environments change, induces the release of oligoribonucleotides. Their release does not kill or injure the bacteria, but rather assists them in establishing a colony in a new environment by increasing their rate of mutation and their rate of growth.
These oligoribonucleotides are released when bacteria enter the pH neutral environment of animals, such as the mouth, nose, oropharyngeal cavity, urethra and vagina.
Through co-evolution, the immune system of animals and humans has adapted a protective response to the appearance of oligoribonucleotides in anticipation of a microbial invasion. This immune reaction is important when oligoribonucleotides (ORNs) are released by bacteria entering the pH neutral body fluids or attempt to invade horizontally onto sterile tissue, (e.g. from the nose to the sinus, from vagina to uterus). Encountering neutral pH will induce the release of these bacterial factors, which will serve to alert the host to a potential penetration onto a sterile area or into sterile tissue. This immune response involves the stimulation of monocytes and macrophages, sentinel cells embedded in oral pharyngeal tissues as well as the B- and T-cells of the immune system.
Specifically, the macrophage has adapted a preemptory reaction to the presence of the oligoribonucleotides (ORNs) that prepares the immune system to defend the host against infection. For example, when ingested bacteria encounter neutral pH or are overcrowded by the presence of growing pathogens, they will release readily absorbable, non-toxic ORNs which activate tissue macrophages to release Interleukin-1, IL-1, Interleukin-6, IL-6 and Tumor Necrosis Factor, alpha, TNFα which stimulate other cells of the immune system. After being highly activated, exposed macrophages down-regulate the surface receptors, CD-14 and CD-16, thereby desensitizing the cell from over-activation by the subsequent interaction with bacterial toxins if infection occurs.
The present inventor has found that the oligomeric fraction having a molecular weight <10 kDa and, in particular, between 500 and 3,000 Da are readily absorbed, are non-toxic, and both activate and modulate the immune system. The products less than 10 kDa in size are non-toxic and contain further a group of compounds of oligomeric size, i.e. 0.5 to 10 kDa that activate and modulate macrophages. Macrophages are activated to release cytokines at levels deemed helpful to combating infections and are down-modulated to prevent their over-activation with the subsequent release of host-threatening levels of cytokines and becoming overly cytotoxic resulting in perforation of organs.
As sentry cells, macrophages circulate in the blood and lymph as well as reside in specialized endothelial tissues and organs. They are among the host's first lines of defense, releasing interleukin signals, destroying microbes and stimulating other immune cells to destroy diseased cells of the host. Approximately twenty different interleukins can be released modifying, amplifying, restricting and dampening messages as the system is stimulated. Thus, the macrophage's signal is key to initiating and enforcing the appropriate immune response. In an infection, bacterial endotoxin (lipopolysaccharide, LPS), binds to the TOLL-like receptor, TLR-4 and the CD-16 surface molecules on macrophages stimulating them and inducing the release of yet higher levels and combinations of IL-1, IL-6 and TNF. These signals, in turn, induce fever, fatigue, cardiovascular hypotension, renal failure and can dictate the death of the host in “septic shock”.
By down-regulating the numbers of CD-14 and CD-16 receptors on the surface of the macrophage, the oligomers released by high levels of stressed bacteria help to ensure that the macrophage does not become over-activated by interleukins thereby creating a system which may go awry killing the host.
Monocytes also respond to the presence of released bacterial ORNs by escaping apoptosis and maturing into macrophages.
Thirteen different species of animal-associated bacteria have been found to release oligoribonucleotides <10 kDa when stressed. However, the distribution of polymer:oligomer:monomer is not equal amongst these species. The levels of ORNs accumulated and released are related to the rate of growth of the bacteria. Therefore, not all strains of bacteria, even of the same species, release levels of oligomers sufficient to protect animals against a subsequent bacterial invasion. An additional discovery is the finding that feral colonies of bacteria yield more oligoribonucleotides (ORNs) when initially stressed than non-feral or laboratory strains. However, inducing a stress upon a laboratory strain before repropagation will stimulate its growth rate and accumulation of ORNs, comparable to that occurring when a feral strain was stressed.
The polymeric fraction (>10 kDa) is toxic when injected into mice, producing a ruffled fur coat, huddling and diarrhea. In vitro assays using human peripheral blood macrophages indicate that the monomeric fraction, (0.5 kDa) does not induce the release of significant levels of interleukins. However, the oligomeric fraction, (between 0.5 and 10. kDa) activates and modulates macrophages, is non-toxic when injected into mice and protects them against a subsequent lethal challenge of injected endotoxin.
The present invention has found that the oligoribonucleotides (ORNs), (between 0.3 and 10. kDa) are a rich new source of natural, normally-occurring, co-evolutionarily evolved immune modulators that can be safely used to protect animals and humans from infections and over-stimulation of their immune system. In addition, this fraction contains compounds that can be used to adjust the expression of individual surface receptors on macrophages to re-center a dysfunctional immune system.
Furthermore, in vitro and in vivo testing indicates the potential role as adjuvants of ORNs <10 kDa by stimulating the production of antibodies. Human B-cells were stimulated in cell culture and mice demonstrated increased B-cell activity when an experimental vaccine against melanoma was injected with ORN<10 kDa.
An unusual feature of the ORNs<10 kDa that possess immune stimulating capacity is their resistance to hydrolysis by RNase (product R-7003 purchased from Sigma Chemical Co.) when used as directed by the manufacturer (incubating 750 μL of ORNs with 50 μg of RNase at 37° C. for 3 hr). The pattern of the ORNs<10 kDa on Sephadex G-10 was the same before and after RNase treatment showing no further reduction in molecular size.
The resistance of ORNs<10 kDa to RNase suggests that the ORNs have unusual structures. Some may have substitutions on their bases (e.g., methyl and other groups) or may have two nucleotide strands held together by complementarity or folded back upon themselves. These unusual structural conditions leading to resistance to RNase are believed to be responsible for their stimulation and modulation of the immune system.
In addition to these unusual structures, the inventor believes that the immune system has also learned through co-evolution to recognize the source of the ORNs<10 kDa by certain features that are unique to the source cell or found to occur at frequencies much higher in one type of cell (e.g., bacteria) than in another (e.g., fungi, animals, etc.). That is to say, the immune system has learned that certain structures reveal the nature of the cellular sources of the ORNs<10 kDa. Two examples of structures specific to microbes are known.
The first are the “signature sequences” of nucleotides known to occur uniquely in the ribosomes of specific orders, families, genera or species of microbes. (Woese, CR, 1998, The Universal Ancestor, Proc Natl Acad Sci 95:6854–9, Olsen, G. J, Woese, C. R, Ribosomal RNA: A Key to Phylogeny, FASEB J. 1993 January; 7(1):113–2, and Zhang, K., Willson, R. C., and Fox, G. E., 2002 Bioinformatics 18(2):244–50 Identification of Characteristic Oligonucleotides in the Bacterial 16S Ribosomal RNA Dataset).
The second are the sequences of nucleotides in DNA that contain the CpG motif in DNA at a much higher frequency in bacteria. Injecting oligodeoxynucleotides (ODN), 4 to 10 nucleotides in length, which contain one or two CpG motifs, have been found to stimulate an immune response. However, they are toxic. (Kreig, A M, et al., 1995, CpG Motifs in Bacterial DNA Trigger Direct B-cell Activation, Nature 374: 546–9).
The inventor believes that the immune system recognizes the ribosomal counterparts of these CpG ODNs as being derived from bacteria. However, through co-evolution, it has adapted a non-toxic, alerting response to their sudden release by invading bacteria.
However <10 kDa ORNs collected from heat-killed bacteria stimulate monocytes as well as those from live bacteria but do not protect mice from endotoxic death. Therefore the Mangan assay previously depended upon cannot be relied upon to predict the results of feeding ORNs to animals. (Mangan D F, Welch G R, Wahl S M, 1991, Lipopolysaccharide, tumor necrosis factor-α, and IL-1B prevent programmed cell death (apoptosis) in human peripheral blood monocytes, J Immunol 146:1541–6).
The discovery of the release of immune-activating and modulating factors has broad implications to improving the immune response through diets and pharmaceutical preparations for humans and animals. Products, (e.g. milk, cheese, yogurts) contain viable bacteria, which, when transferred to the nutrient deprived environment of pH neutral body fluids, such as the mouth, release ORNs. If such products were formulated to extend the dwell-time in the mouth and throat, more ORNs would be released, activating and modulating a greater immune response. Likewise, gels used to administer probiotics would deliver more ORNs if the pH of the gel were buffered to neutral pH rather than acidic.
Numerous patents teach the healthful benefits of administering specific viable bacteria to humans and animals either orally or parenterally to provide local immune stimulation. Additionally, the prior art recognizes the importance of modulating interleukin release but does not teach the use of safe, natural, normally occurring products derived from co-evolution, which are effective when taken orally. However, the present invention teaches the administration of sterile, stable, controlled doses of the active principle, ORNs, rather than unstable, viable microorganisms.
U.S. Pat. No. 4,975,467 teaches methods by which synthetic compounds can be used to inhibit the release of IL-1 thereby alleviating the induction of its pathophysiologic conditions. U.S. Pat. No. 5,055,447 provides methods and compositions for the prevention of septic shock by administering growth factor-β. This patent teaches the use of administering a signal compound to intercept or modify existing signals. U.S. Pat. Nos. 5,041,427 and 5,158,939 teach the use of a non-toxic LPS from R. spaeroides, ATCC 17023 to desensitize macrophages to toxic LPS. Since R. spaeroides has an unusual lipid A structure, it may not be effective as a desensitizing agent. U.S. Pat. No. 5,157,039 supports the clinical importance for controlling IL-1 release by macrophages by teaching the use of two non-natural quinolinol compounds, which appear to be non-selective in IL inhibition. U.S. Pat. No. 5,840,318 teaches that bacteria exposed to a neutral pH buffer for 10–16 hours release compounds <10 kDa in size having a UV maximum absorption at 254 nm. Feeding the <10 kDa sizes activates and modulates an animal's immune system, thereby protecting it against a subsequent lethal challenge of endotoxin. The accumulation of A-254 absorbing compounds is associated with the slowing or termination of bacterial cell growth. Further, U.S. Pat. No. 6,589,771 B1 teaches that bacteria enter dormancy during their stationary phase by accumulating solutes that increase the osmolality and H+ that decrease the intracellular pH of the cell. Putting dormant cells in buffers with osmolalities and hydrogen ion concentrations lower than that of the cell will cause the diffusion of solutes and H+ thereby inducing the arousal of the cell. Rather than a single 10–16 hour wash to induce the release of the compounds that have a maximum of UV absorption at 254 nm, one can employ a series of 20-minute washings in a buffer of pH 7 and of lower osmolality followed by an adjustment period of approximately 72 hours in the final wash.
Livestock are routinely fed silage, a fermented product containing high levels of viable harmless bacteria. When ingested and chewed as cud, the silage bacteria release immune-activating ORNs. The proper selection of harmless bacteria that ferment silage and grains and also release significant levels of ORNs will help improve the health of livestock.
Direct-fed microbials and probiotics are harmless bacteria which are grown in a rich media, concentrated, dried and fed to animals either as a powder for top-dressing or in gel forms for oral inoculation. These products provide a health benefit to the animal in combating infections relating to shipping and weaning. Analysis of commercial products indicates the presence of ORNs as well as the release of ORNs when transferred to saliva or to a nutrient-reduced environment. It is believed the presence and release of ORNs explains their effectiveness. This discovery permits the administration of a sterile, stable, probiotic of known dose for livestock and poultry. Currently, viability of probiotics is believed necessary for effectiveness. A sterile, stable product allows distribution without refrigeration and would provide a known dose.
The present inventor has now found that the extrusion of ORNs from the cell is induced when cells are exposed to neutral aqueous environments, e.g. saliva, body fluids and physiologic saline. Extrusion can be induced by subjecting the bacterial populations to repeated dilutions or washings, as many as 6, in pH neutral buffers of sufficient molarity to offset the efflux of H+ that accompany ORNs. Further, they have discovered doses of ORNs that are also effective in rescuing monocytes from apoptosis and, in addition, rescue animals from the lethality of endotoxemia. Moreover, lab tests have been developed which aid in the selection and screening of bacteria which release the more potent ORNs and determining appropriate doses.
It is therefore an object of this invention is to provide a method to capture the accumulated ORNs released when bacteria enter a pH neutral buffer and administer a sufficient quantity of bacteria-free <10 kDa to animals for the purpose of stimulating an immune response that protects against subsequent microbial infections.
A further object of this invention is to provide oral pharmaceutical compositions to help prevent microbial infections in animals.
Another object of this invention is to provide parenteral injections of individual components of these pharmaceutical compositions to treat endotoxic shock.
A further object of this invention is to provide topical pharmaceutical compositions for the activation and modulation of local immune systems to protect against ear, nose and vaginal infections.
An additional object of this invention is to use individual components of these pharmaceutical compositions as adjuvants in conjunction with vaccination.
A further object of this invention is to provide pharmaceutical compositions to down regulate the cytotoxicity of macrophages and prevent their destruction of normal T-cells in persons suffering from HIV infections.
It is still a further object of this invention to provide ORNs to extend the viability of monocytes, thereby improving their ability to mature into macrophages to fight infection.
It a further object of this invention to provide methods to ensure that bacteria will accumulate and release higher levels and more potent ORNs.
These and other objectives will become apparent from the following detailed description of the invention. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.